A fundamental difference in sugar metabolism exists between slow-growing tumor cells found in the hypoxic regions of most solid tumors and the majority of normal cells in the body, which are under oxygen. Hypoxic cells rely solely on glycolysis for energy production, whereas cells under normal oxygen tension can metabolize other carbon sources through oxidative phosphorylation. This creates a natural window of selectivity that can be exploited for therapy by using inhibitors of glycolysis. Based on our in vitro and in vivo results, a Phase I clinical trial was recently initiated to test the hypothesis that the glycolytic inhibitor, 2-deoxyglucose (2-DG), which targets the most resistant cell population found in solid tumors, slowly-growing hypoxic cells, will raise the efficacy of treatment when combined with standard chemotherapy which targets the rapidly-dividing aerobic tumor cells. The direction for the continuation of this competitive renewal grant stems from three recent findings: The first is that the glucose analog, 2-fluro-deoxyglucose (2-FG), which is used to locate and identify tumors in patients by PET scan, has been found to be 3x more potent than 2-DG in inhibiting glycolysis and killing hypoxic tumor cells in our in vitro models. Thus, Aim #1 is directed at determining whether 2-FG has better activity than 2-DG in killing hypoxic cells in vivo. The second finding is that the ubiquitous hypoxia-inducible factor (HIF) mediates resistance to glycolytic inhibitors in hypoxic tumor cells. Results from Aim #1 will be combined with in vitro experiments in Aim #2, which are geared toward understanding how hypoxic tumor cells become resistant to glycolytic inhibitors thru up-regulation HIF. The third recent finding is that a select number of tumor cell types growing in the presence of oxygen are killed by 2-DG but not by 2-FG. Based on data from the 1970's in which 2-DG was shown to interfere with N-linked glycosylation of viral coats, it appears that the toxicity we find in these select tumor cells with 2-DG is due to the same mechanism (as opposed to inhibition of glycolysis) which forms the focus of Aim #3. Thus, the long-term goals of this proposal, which are directly relevant to public health are the following: (1) To generate data that will eventually lead to the use of 2-FG as a more potent inhibitor of glycolysis in the clinic;(2) To improve the efficacy of glycolytic inhibitors in patients by combining them with anti-HIF agents;and (3) To provide a rational basis for the clinical use of 2-DG as a single agent to kill both aerobic (via interference with glycosylation) and hypoxic (via blockage of glycolysis) cell populations in select tumor cell types that are identified in vitro to be sensitive to this sugar analog in the presence of oxygen.